Inhibition of Quorum Sensing and Biofilm Formation in Pathogenic Bacteria by Euphorbiaceae-Derived Phytochemicals Purified Through Column Chromatography

Abstract

Bacteria use communication systems known as quorum sensing (QS) to coordinate group behaviors such as toxin production and biofilm formation. These processes make infections harder to treat and contribute to antimicrobial resistance. With the rise of multidrug?resistant (MDR) infections, there is an urgent need for new ways to weaken pathogens without necessarily killing them directly. Plant?derived compounds are attracting attention because they often contain complex chemical structures that can interfere with microbial communication. Members of the Euphorbiaceae family, long valued in traditional medicine, are rich in flavonoids, terpenoids, and phenolic compounds that show promise as antimicrobial agents. In this study, phytochemicals were extracted from selected Euphorbiaceous plants and purified using column chromatography. Fractions were analyzed with thin?layer chromatography (TLC), Fourier?transform infrared spectroscopy (FTIR), and high?performance liquid chromatography (HPLC) to identify active compounds. Their activity was then tested against common clinical pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. To assess effects on QS, the violacein assay was carried out with Photobacteriumviolaceus CV026. Biofilm inhibition was measured using crystal violet staining and further visualized with confocal laser scanning microscopy. The results showed that fractions enriched in flavonoids and terpenoids strongly reduced violacein production and disrupted biofilm formation, yet did not significantly inhibit bacterial growth. This indicates that their action is directed at bacterial signaling rather than direct killing. Fractions from Euphorbia Herta and Phyllanthus amaro’s were particularly effective, with up to 70% biofilm reduction in P. aeruginosa. Microscopy confirmed a clear breakdown of biofilm structure in treated samples. Hydroxylated flavonoids appeared to play a central role in this effect. These findings highlight the potential of Euphorbiaceous phytochemicals as natural ant virulence agents. By targeting communication and cooperation within bacterial populations, they offer a strategy that may slow down the development of resistance while supporting current antibiotics. This work supports further exploration of Euphorbiaceous compounds, including more detailed molecular studies and in vivo testing, to develop new tools against persistent and resistant infections.

Introduction

1. Background and Rationale

• Antimicrobial resistance (AMR) is a major global health crisis, driven by overuse and misuse of antibiotics.

• Traditional antibiotics are becoming ineffective, especially against biofilm-forming bacteria like Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

• Biofilms protect bacteria from antibiotics and immune responses, making infections chronic and recurrent.

• A promising alternative strategy involves disrupting quorum sensing (QS) — the bacterial communication system that regulates virulence and biofilm formation.

2. Quorum Sensing (QS)

• QS enables bacteria to sense population density and coordinate gene expression using autoinducers.

• Gram-negative bacteria (e.g., P. aeruginosa) use AHLs, while Gram-positive bacteria (e.g., S. aureus) use peptides.

• QS controls biofilm formation, making it a key target in antimicrobial strategies.

3. Role of Euphorbiaceous Plants

• Plants from the Euphorbiaceous family, such as Euphorbia herta and Phyllanthus amarus, have traditional medicinal use and demonstrated antimicrobial, anti-inflammatory, and QS-inhibitory properties.

• Their rich content of flavonoids, terpenoids, alkaloids, and tannins makes them effective candidates for disrupting QS and biofilms.

4. Research Objective

• The study aimed to evaluate purified phytochemical fractions from Euphorbiaceous plants for:

  • Quorum sensing inhibition

  • Biofilm disruption

  • Non-bactericidal virulence attenuation (reducing resistance development)

5. Methodology Overview

• Plant Collection & Extraction: Methanol extraction via Soxhlet; fractions separated by polarity (hexane, chloroform, ethyl acetate, butanol, aqueous).

• Purification: Column chromatography followed by TLC, FTIR, and HPLC for compound identification.

• Microbial Assays:

  • QS inhibition: Using Chromobacterium violaceum CV026 (violacein assay).

  • Biofilm assay: Crystal violet staining and confocal microscopy for visual confirmation.

• Bacterial strains: Clinical isolates of P. aeruginosa, S. aureus, and E. coli.

6. Key Findings

A. Quorum Sensing Inhibition

• QS inhibition was dose-dependent.

• At 500 µg/mL:

  • P. aeruginosa showed ~90% inhibition.

  • S. aureus: ~75%

  • E. coli: ~68%

• P. aeruginosa was most susceptible, likely due to its well-defined QS systems.

B. Biofilm Inhibition

• Methanol and ethyl acetate fractions were most effective:

  • P. aeruginosa: >80% inhibition

  • S. aureus: ~70–75%

  • E. coli: ~55–60%

• Aqueous fractions were least effective.

C. Microscopy and Visual Evidence

• Confocal microscopy revealed:

  • Treated biofilms had sparse bacteria and reduced EPS matrix.

  • Structural disruption aligned with reduced QS activity.

7. Discussion and Implications

• Euphorbiaceous phytochemicals interfere with QS, reduce biofilm formation, and weaken bacterial virulence without directly killing the microbes.

• This anti-virulence approach minimizes the selective pressure for resistance.

• The effectiveness of semi-polar fractions supports the role of flavonoids and polyphenols in disrupting bacterial communication and biofilm integrity.

• Integrating traditional medicinal knowledge with modern techniques strengthens the case for plant-based QSIs as viable therapeutic agents.

Conclusion

The present study highlights the antimicrobial potential of phytochemical fractions derived from Euphorbiaceous plants, with a particular focus on their ability to inhibit quorum sensing (QS) and biofilm formation in clinically relevant pathogens. Through systematic extraction, purification, and characterization, the fractions demonstrated significant biological activity, especially against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. The quorum sensing inhibition assays revealed a strong concentration-dependent effect, with P. aeruginosa exhibiting the highest sensitivity to Euphorbiaceous fractions. This finding underscores the potential of plant-derived compounds to target communication pathways that regulate bacterial virulence. Since QS plays a critical role in the production of toxins, enzymes, and other pathogenic determinants, its disruption provides an effective strategy for attenuating infection without necessarily inducing selective resistance pressure. In parallel, the biofilm inhibition assays confirmed that suppression of QS correlates with weakened biofilm architecture. Methanolic and ethyl acetate fractions exhibited the strongest inhibitory effects, suggesting that semi-polar phytochemicals such as flavonoids, tannins, and phenolic acids are likely responsible for the observed activity. In contrast, aqueous fractions showed weaker results, indicating lower concentrations of active metabolites. These outcomes emphasize the importance of solvent choice in maximizing the yield of bioactive compounds during extraction. The schematic and workflow figures further strengthened the findings by providing a visual representation of both the methodological approach and the structural effects on bacterial biofilms. Together, the data support the conclusion that Euphorbiaceous-derived phytochemicals can effectively disrupt EPS synthesis and bacterial clustering, thereby destabilizing mature biofilms.

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Copyright

Copyright © 2025 Dr. Annu Tiwari, Dr. Amit Tiwari. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.